Gliomas are the commonest intra-axial brain tumors generally divided into two groups for practical purposes: low-grade (grades I and II) and high-grade (grades III and IV) gliomas [
On the other hand, the most common extra-axial tumors in the intracranial compartment are meningiomas, originated from the cap cells of arachnoid. These tumors were classified by the World Health Organization into three grades (grades from I through III); grade III is the malignant form and recurrence is very high [
Since we do not have still curative treatment with respect to many solid cancers, identifying a specific marker or a target becomes vital or understanding the molecular basis behind these tumors may lead us to developed curative treatment. The rapidly expanding data on endothelial cell-specific molecule-1 (ESM-1) or endocan shows that it is a soluble dermatan sulfate proteoglycan (PG), freely circulating molecule in the blood, and present primarily on the cell surface, in the extracellular matrix and body fluids [
This study was conducted in Istanbul University, Cerrahpasa Medical Faculty and Departments of Neurosurgery and Biochemistry, during the year 2015. All patients or next of kin were fully informed and ethical approval for this study was obtained from the Human Investigations Committee at Istanbul University. We excluded the patients who had any kind of chronic or acute infection, immunological and metabolic diseases, neoplastic disease of other organ systems, any cardiovascular diseases, and recent major surgical procedure at the time of tumoral tissue collection, in which endocan status might be affected.
A total of 59 adult patients and 15 controls served as subjects in this study. The tumor groups were divided into three groups:
A total of 74 tissue samples were assayed for endocan. For each patient, tumor tissues were collected during surgery, and from the control group, brain tissues were obtained during the autopsy procedure. Brain tissues from the controls were obtained within the first 4 h following death. As soon as possible, each sample was stored at −80°C until being assayed.
Brain tumor and cadaver brain tissue samples were washed in cooled 0.9% NaCl and placed on an ice-cold plate, incised, and weighed. The samples were then immediately frozen in liquid nitrogen until they were homogenized. Tissue samples were homogenized manually in homogenizing buffer (100 mM KH2PO4–K2HPO4), to obtain 20% homogenates, with a tissue grinder fitted with a Teflon pestle for the measurement of adhesion molecules’ levels and for total protein determination. The homogenates were sonicated with MSE sonicator two times at 30 sec intervals on ice, with a power output of 38 watts. Supernatant fractions were obtained by the centrifugation of the homogenates in 15000 ×g for 15 min. During aliquot preparation, supernatant fractions were maintained at +4°C in dim light. The supernatant fractions were divided into aliquots (one for each assay) and immediately stored at −80°C (for 2 weeks maximum) for the measurement of biochemical parameters.
Endocan levels were measured with commercially available enzyme linked immunosorbent assay (ELISA) kits (YH Biosearch Laboratory, Shanghai, China) based on biotin double antibody sandwich technology. Actual levels of this parameter in the samples were determined from the standard curves.
Total protein content of the samples was measured by using the modified method of biuret with some volumetric modifications, proposed by Itzhaki and Gill [
We used a commercially available statistical software package (SPSS version 14.0 Inc., Chicago, IL, USA) for all the statistical analyses. The mean ± standard deviations (±SD) were calculated for each parameter. For all comparisons, the nonparametric Mann-Whitney
Statistical results including the mean (±SD) and probability (
The mean (±SD) value and results of statistical comparisons of endothelial cell-specific molecule-1 between the groups.
Parameters/groups | Controls | Meningioma | LGG | HGG |
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ESM-1 (ng/mg protein) | 9.66 ± 1.45 | 11.8 ± 1.62 | 13.9 ± 2.90 | 16.1 ± 2.59 | |
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Controls versus meningioma | 0.001 | ||||
Controls versus low-grade gliomas | 0.0001 | ||||
Controls versus high-grade gliomas | 0.0001 | ||||
Meningioma versus low-grade gliomas | 0.01 | ||||
Meningioma versus high-grade gliomas | 0.0001 | ||||
Low-grade gliomas versus high-grade gliomas | 0.02 |
C: controls; ESM-1: endothelial cell-specific molecule-1; HGG: high-grade glioma; LGG: low-grade glioma; M: meningioma.
Overall survival and endocan levels (ng/mg protein) in patients studied here.
Number | Endocan levels in the groups | Follow-up (months) | |||
---|---|---|---|---|---|
Controls | MGs | LGGs | HGGs | ||
1 | 8.12 | 12.27 | 14.55 | 15.62 | 36/ |
2 | 9.76 | 14.50 | 12.65 | 19.05 | 36/ |
3 | 11.08 | 12.98 | 13.20 | 22.30 | 12/ |
4 | 10.91 | 11.62 | 18.90 | 17.50 | 36/ |
5 | 10.72 | 12.02 | 11.77 | 14.36 | 16/ |
6 | 7.92 | 10.91 | 12.27 | 12.78 | 60/ |
7 | 8.41 | 9.23 | 10.78 | 18.90 | 24/ |
8 | 8.77 | 11.77 | 12.98 | 14.55 | 36/ |
9 | 11.20 | 10.72 | 9.25 | 16.71 | 36/ |
10 | 10.32 | 9.21 | 17.62 | 17.62 | 36/ |
11 | 8.41 | 11.21 | 10.91 | 15.02 | 36/ |
12 | 7.65 | 12.36 | 9.23 | 16.71 | 23/ |
13 | 12.04 | 14.32 | 14.52 | 12.65 | 13/ |
14 | 8.62 | 10.27 | 14.60 | 10.91 | 23/ |
15 | 11.08 | 11.41 | 17.30 | 18.09 | 12/ |
16 | — | 14.55 | 14.50 | 17.52 | 13/ |
17 | — | 14.36 | 18.62 | 15.94 | 24/ |
18 | — | 12.50 | 16.02 | 15.94 | 36/ |
19 | — | 10.91 | 14.50 | 14.20 | 12/ |
20 | — | 10.65 | — | 17.60 | 24/—/18 |
D: died; HGGs: high-grade gliomas; LGGs: low-grade gliomas; MGs: meningiomas.
Mean level of endocan showed a steady incline from the controls to the most malignant form of the tumor groups. All the tumor groups showed significant higher levels compared to the controls (
While this paper was being written, mean follow-up times for meningiomas, LGGs, and HGGs were found to be
There was no death in LGGs group and 3 showed unremarkable progression of the residue which was decided to be followed up radiologically in 3 months’ interval. No patient in this group received radio- or chemotherapy. Four patients showed seizure which was present since surgery and they were on antiepileptic medication.
As expected, the most dramatic changes were noted in HGGs group. All received radio- and chemotherapy. Eleven (84.6%) of the thirteen patients with grade IV glial tumor died during the follow-up period. The two patients who are still alive showed no radiological recurrence and are free of clinical symptoms. Seven patients diagnosed with grade III glial tumors are still under our follow-up and all patients are alive and are still on antiepileptic medication. Table
Correlation analysis between endocan levels and overall survival within the tumor groups showed negative relationship (
It has been demonstrated that endocan is expressed from endothelium of vascularized organs but surprisingly highly vascularized organs such as heart, pancreas, liver, and brain do not contain endocan [
The current study is the first prospective study to show expression of endocan in common brain tumors, namely, gliomas and meningiomas. No study noted so far showed endocan expressions in meningioma. Previous studies showed that endocan expression is not noted in normal brain tissue although it is one of the highly vascularized organs [
The authors want to acknowledge some limitations in this study. The first limitation is that we included small sample size of the patients in each group. This is because we included consecutive patients who gave permission to us and because of other strict inclusion criteria. We especially wanted to see whether endocan levels increased on common brain tissues and especially in meningiomas and LGGs in which results are preliminary. Thus, in spite of small sample size, this study may give an idea to the neurosurgeons who want to develop further studies which should include larger sample size of patients. Second limitation is that we did not perform immunohistochemistry studies on tumor tissues which would be more validated and further studies on brain tumors should be designed to include immunohistochemistry and the third limitation in this study is that serum levels were not studied. Our next study will include LGGs in which we will measure serum levels of endocan before and after surgery to see whether there is any correlation between serum levels of endocan and surgical removal. In conclusion, depending on the current literature and our own findings, endocan may be a marker of tumor aggressiveness in common brain tumors: as the grade of the tumor increases, endocan level increases. Since studies including brain tumors are very limited, further studies are needed to verify our findings and future results may lead us to develop monoclonal antibody directed to endocan to prevent aggressiveness of the brain tumors.
The tumor and control samples here were reused for the extension of our previous works.
The authors in this study declare no conflict of interests.
This work was supported by “Scientific Research Projects Coordination Unit” of Istanbul University (Project no. 55673).